New or improved tools to gain a better understanding of how neurons process information and modulate activity are critical. Because the principal function of a neuron is to process electrical signals, one tool that is most in need for improvement is membrane potential imaging with voltage-sensitive probes (Vm imaging). This application seeks to build a high speed imaging system that will have the sensitivity to cover the full range of spatial and temporal resolutions required in monitoring neuronal activity in brain slices. Presently, such a system is not in existence. The spatial resolution will range from recording Vm-signals from individual dendritic spines to monitoring simultaneously activity of large ensembles of neurons. Subcellular, single spine resolution in Vm-imaging has been attempted (Nuriya et al, 2006) but never satisfactory achieved. Single spine resolution is, however, essential because excitatory synapses on individual dendritic spines are elementary units underlying mechanisms of short and long term plasticity. At the same time, cellular mechanisms of plasticity must be integrated in the context of organizational and integrative aspects of persistent synaptic plasticity in different brain region at the macroscopic level. Monitoring large neuronal ensembles is needed to provide spatial maps of plasticity expression over large areas of the brain circuitry. In order to accurately monitor action potential (AP) signals, high-temporal resolution of 2,000 - 10,000 frames per second will be provided by utilizing a high-speed CCD camera. The basic principles governing the sensitivity of high-speed, high-spatial resolution Vm- imaging indicate how proposed improvements can be implemented. Also, all necessary components for the proposed system are available and have been tested and demonstrated to be functional (preliminary experiments). The imaging system will use a frequency-doubled diode-pumped Nd:YVO4 at 532 nm as an excitation source in wide-field epi-fluorescence microscopy and a high-speed CCD camera for acquisition. An additional aim of this proposal is to further develop and test new voltage-sensitive probes and protocols for selectively labeling the plasma membrane of individual neurons with minimal intracellular labeling in order to achieve the maximum signal-to-noise (S/N). PUBLIC HEALTH RELEVANCE: The research on normal synaptic function and plasticity in the nervous system seeks fundamental insights into the mechanisms by which neural circuits control behavior. Through these insights, it may become possible to clarify the root causes of diseases that affect millions, including schizophrenia, mood disorders, degenerative brain disorders, epilepsy, and neurodevelopmental disorders such as autism.